Recently we developed a new type of RNA nanostructure that forms a truncated tetrahedron. The structure was built from our hexameric ring where 4 sides of the tetrahedral structure each contain the hexmeric ring, but each ring contains 3 H-shaped crossover connectors to the other rings. This type of construct allows for the incorporation of up to 12 functional entities such as Dicer substrates, beacons and/or aptamers. We found that cells seem to take up these constructs better than some of the other RNA nanoconstructs. The hypothesis that nanoparticle shape and size matter regarding functionality seems to be true. Due, at least in part, to the better uptake we found that knockdown of targeted genes to induce cell death, using incorporated Dicer substrate PLK1 is more efficacious than some of our other particles. Several different methods were used to verify the assembly of this particle including the newly acquired atomic force microscope (AFM). Currently we are also collaborating with the Cryo-EM core to further characterize these particles.---Previously, programmable hexameric RNA rings were developed for the controlled delivery of up to six different functionalities. To increase the potential for functionalization with little impact on nanoparticle topology, we introduced gaps into the double-stranded regions of the RNA rings. Molecular dynamics simulations were used to assess the dynamic behavior and the changes in the flexibility of the designs. The changes suggested by simulations, however, cannot be clearly confirmed by conventional techniques such as nondenaturing polyacrylamide gel electrophoresis and dynamic light scattering. Also, an in vitro analysis in primary cultures of human peripheral blood mononuclear cells does not reveal any discrepancy in the immunological recognition of the new assemblies. To address these deficiencies, we introduced a computer-assisted quantification strategy, which is based on an algorithmic AFM-resolved deformation analysis of the RNA nanoparticles studied on a mica/air interface. We validated this computational method by manual image analysis and fitting it to the simulation-predicted results. The presented nanoparticle modification strategy and subsequent AFM-based analysis provided a broad-spectrum approach for the future development of nucleic acid-based nanotechnology. ----Typical methodologies that utilize RNAs as targeting agents to control diseased cells rely on pre-analysis of the state of the cells to be targeted followed by delivery of an agent e.g. antisense, antimiR or siRNA, thus separating the therapeutic step and the diagnonostic step. We developed a set of context-sensitive RNA-based logic switches that combines both steps into one logic system. This permits the the conditional activation or deactivation of the the release of single-stranded or double-stranded RNAs as a function of expressed RNAs. THe switches are designed using RNA/DNA hybrid significantly limiting issues related to nuclease degredation. ---To achieve control over deliverable functionality and stability of RNA-based nanoparticles, the properties of DNA and RNA were merged in the development of computationally designed nanoparticles that were constructed from RNA/DNA hybrids. These molecules allow higher stability in blood serum, attachment of fluorescent markers for tracking, and the ability to split the components of functional elements inactivating them, but allowing later activation under the control of complementary toeholds by which the kinetics of re-association can be tuned. Diceable substrate siRNA could be split into two components, each consisting of an RNA/DNA hybrid. Complementary RNA single-stranded toeholds rather than DNA can be used in the construction of the hybrids. The two hybrids, when transfected into cells recombine into two products due to the toeholds and the computationally determined thermodynamic difference between the hybrids and the products. From the perspective of thermodynamics, the use of RNA toeholds is advantageous as it reduces the length of the single stranded ends required to unzip the hybrids and generate the functional RNA element. From a design perspective, the RNA toehold can be part of the functional DS RNA, or other potential RNA moiety, reducing the size and minimizing the design constraints of the resulting hybrid duplexes. RNA-based hybrids containing 3 Dicer substrate siRNAs for synergistic simultaneous targeting of apoptosis-related genes in HT29 tumors are now being used, after significant testing in cell cultures, in a comprehensive mouse study funded, in part, by the Invention Development Program. Initial results look encouraging showing retardation of tumor growth both intratumorally and more so by tail vein injection. Further studies are being performed using alternative delivery agents. ----Since we can control immune response with RNA-based nanoparticles, we have been collaborating with Joost Oppenheim- CCR, and Chris Jewell-UMD to take advantage of these properties to activate the immune system for anti-cancer treatment (funded in part by an UMD-NCI Partnership for Integrative Cancer Research grant). Working with Joost Oppenheim group we found possibly significant in vivo results showing a cure in 3 out 10 immune competent mice. Most of the other mice showed signficant regression ot their tumors. Further experiments are planned to characterize more fully the pathways of action. ---The delivery of RNA-based nanoconstructs in cell culture and in vivo is essential for the development of therapeutic methodologies using these agents. Non-modified naked RNAs have short half-lives in blood serum due to nucleases and have difficulty crossing cell membranes due to their negative charge. Thus, we are developing lipid and polymer formulations. In the case of the lipids we have constructed delivery agents consisting of DOTAP, DOPE and DSPE-PEG2000 to target cancer cells (in collaboration with Esta Sterneck, CCR). Experiments look quite positive. In addition, we are working with Jonathan Lovell (U of Buffalo) on the development of photoactivatable polymers for the delivery of our RNA-based nanoparticles. Results are very encouraging here too, showing minimal leakage without laser treatment and significant functionality when laser treated. A second-generation chlorin-based photosensitizer, HPPH shows tremendous therapeutic potential in clinical trials in treatment of esophageal cancer. We, in collaboration with Sunil Dubey (Birla Institute of Technology & Science) have developed and validated a bioanalytical method for estimation of HPPH (a compound used in photodynamic therapy) in rat plasma using High Performance Liquid Chromatography with PDA detector. --Colon-26 mice using an HPPH LNP showed superior efficacy using PDT. We have also tested bolaamphiphile vesicles GLH-19 and GLH-20 formulations for delivery of siRNA to tumors and to the brain. We showed good delivery to both locations, including the brain which is difficult to target due to issues related to crossing the blood-brain barrier. The stability of the various formulations tested were also analyzed by molecular dynamics, which explained quite well the results we were seeing experimentally.---An exclusive license of two of our patents was established with a startup company that was established out of the NCI Nanochallenge. The plan is for the company is to use our RNA-based nanoparticles for glioblastoma.